DE19609479A1 - Endotoxin-specific membranes - Google Patents
Endotoxin-specific membranesInfo
- Publication number
- DE19609479A1 DE19609479A1 DE19609479A DE19609479A DE19609479A1 DE 19609479 A1 DE19609479 A1 DE 19609479A1 DE 19609479 A DE19609479 A DE 19609479A DE 19609479 A DE19609479 A DE 19609479A DE 19609479 A1 DE19609479 A1 DE 19609479A1
- Authority
- DE
- Germany
- Prior art keywords
- endotoxin
- microfiltration membrane
- membrane according
- spacer
- endotoxins
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
- B01D67/00931—Chemical modification by introduction of specific groups after membrane formation, e.g. by grafting
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
- A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods
- A61L2/0017—Filtration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
- A61L2/022—Filtration
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
- B01D67/0081—After-treatment of organic or inorganic membranes
- B01D67/0093—Chemical modification
- B01D67/00933—Chemical modification by addition of a layer chemically bonded to the membrane
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
- B01D69/14—Dynamic membranes
- B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/26—Synthetic macromolecular compounds
- B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
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- B01J20/28002—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their physical properties
- B01J20/28004—Sorbent size or size distribution, e.g. particle size
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- B01J20/28033—Membrane, sheet, cloth, pad, lamellar or mat
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- B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
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- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
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- B01J20/3251—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising at least two different types of heteroatoms selected from nitrogen, oxygen or sulphur
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- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
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- B01J20/3248—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such
- B01J20/3255—Non-macromolecular compounds having a well defined chemical structure the functional group or the linking, spacer or anchoring group as a whole comprising at least one type of heteroatom selected from a nitrogen, oxygen or sulfur, these atoms not being part of the carrier as such comprising a cyclic structure containing at least one of the heteroatoms nitrogen, oxygen or sulfur, e.g. heterocyclic or heteroaromatic structures
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Abstract
Description
Die Erfindung betrifft eine Mikrofiltrationsmembran zur Abtren nung von Endotoxinen aus flüssigen Medien sowie die Verwendung dieser Mikrofiltrationsmembran.The invention relates to a microfiltration membrane for separation endotoxins from liquid media and their use this microfiltration membrane.
Endotoxine sind Lipopolysaccharide aus der äußeren Zellmembran Gram-negativer Bakterien, die als Pyrogene wirken. Aufgrund der Allgegenwärtigkeit von Bakterien sind auch Endotoxine ubiquitär. Sie können im Gegensatz zu Bakterien jedoch nicht durch Stan dard-Methoden wie Sterilfiltrieren oder Autoklavieren entfernt bzw. unschädlich gemacht werden (1). Aus diesem Grund ist steril nicht gleichbedeutend mit Endotoxin-frei. Besonders kritisch ist die Anwesenheit von Endotoxinen in Injektions- bzw. Infusionslö sungen (Parenteralia), da sie intravenös appliziert bereits in Mengen von 1 ng pro kg Körpergewicht fiebererregend wirken. Die Symptomatik reicht bei entsprechend hoher Dosierung (z. B. durch großvolumige Parenteralia) bis zu schwerem Schock und Tod (2, 3). Daher schreiben fast alle Pharmacopöen neben der Keimfrei heit strenge Endotoxin-Höchstwerte vor: z. B. 0,2 EU pro mg Chloramphenicol zur Injektion oder nur 0,003 EU pro Heparin-Unit (4). Die Erfüllung dieser Forderungen bereitet in der Praxis einige Schwierigkeiten. Insbesondere die Produktion biologischer Arzneimittel kann nicht in allen Schritten Endotoxin-frei erfol gen. Als Endotoxin-Quellen kommen hauptsächlich in Frage:Endotoxins are lipopolysaccharides from the outer cell membrane Gram-negative bacteria that act as pyrogens. Due to the The ubiquity of bacteria is ubiquitous, too. However, unlike bacteria, you cannot use Stan Standard methods such as sterile filtration or autoclaving are removed or made harmless (1). For this reason it is sterile not synonymous with endotoxin-free. Is particularly critical the presence of endotoxins in injection or infusion solution solutions (parenterals) since they are already administered intravenously in Amounts of 1 ng per kg body weight have a febrile effect. The Symptoms are sufficient with a correspondingly high dosage (e.g. through large volume parenterals) to severe shock and death (2, 3). Therefore, almost all pharmacopoeias write next to the aseptic strict endotoxin levels: e.g. B. 0.2 EU per mg Chloramphenicol for injection or just 0.003 EU per heparin unit (4). In practice, these requirements are met some difficulties. In particular, the production of organic Medicinal product cannot be endotoxin-free in all steps The main possible sources of endotoxin are:
- - Rohstoffe wie Plasma oder Gewebe, die bereits Bakterien-konta miniert sein können.- Raw materials such as plasma or tissue that already contain bacteria can be mined.
- - Bei rekombinanten Produkten ist mit dem Eintrag von Host-spe zifischen Endotoxin zu rechnen.- In the case of recombinant products, the entry of host-spe specific endotoxin.
- - Bakterielle Kontamination von Geräten, Filtern oder Hilfsstof fen während der Herstellung.- Bacterial contamination of devices, filters or auxiliary substances fen during manufacture.
Die für thermostabile Wirkstoffe übliche Hitzedekontamination (30 Minuten bei 250°C) ist für die Präparate ebenso wenig ge eignet wie Behandlung mit Säuren, Laugen oder stark oxidierenden Agenzien (H₂O₂) (1).The usual heat decontamination for thermostable active ingredients (30 minutes at 250 ° C) is also not ge for the preparations suitable as treatment with acids, bases or strongly oxidizing Agents (H₂O₂) (1).
Beim Einsatz von Aktivkohle oder Tiefenfiltern wie ZETA PLUS sind häufig merkliche Produktverluste zu verzeichnen, so daß ihre Anwendung auf die Wasseraufbereitung beschränkt bleibt (5, 6).When using activated carbon or depth filters such as ZETA PLUS there are often noticeable product losses, so that their application remains limited to water treatment (5, 6).
Die Ultrafiltration hat als sehr schonende Methode große Popula rität auf dem Gebiet der Endotoxin-Entfernung erlangt. Hierbei wird i. a. mit cut-offs von 10 000 oder 5000 gearbeitet, um auch monomere Bestandteile (MW ca. 14 000) wirkungsvoll abzutrennen, die neben hochmolekularen Aggregaten (bis zu mehreren Millionen Molekulargewicht) vorliegen. Trotzdem treten immer wieder Pro bleme mit niedermolekularen Spaltstücken auf, die ebenfalls pyrogen wirken (z. B. Lipid A). Das betrifft vor allem die Hämo dialyse: Obwohl die Dialysepuffer ultrafiltriert werden, entwickeln z. B in den USA jährlich 400 000 Hämodialyse-Patienten septische Symptome (7). - Die Notwendigkeit der kleinen cut-offs beschränkt die Anwendung der Ultrafiltration zudem auf die De kontaminierung niedermolekularer Substanzen (8).Ultrafiltration has a large population as a very gentle method achieved in the field of endotoxin removal. Here will i. a. worked with cut-offs of 10,000 or 5,000 to also effectively separate monomer components (MW approx. 14,000), which in addition to high molecular weight aggregates (up to several million Molecular weight). Nonetheless, there are always pros bleme with low molecular weight fragments, which also have a pyrogenic effect (e.g. Lipid A). This mainly affects hemo dialysis: although the dialysis buffers are ultrafiltered, develop e.g. B 400,000 hemodialysis patients annually in the United States septic symptoms (7). - The need for small cut-offs also limits the use of ultrafiltration to De contamination of low molecular weight substances (8).
Im Fall von hochmolekularen Produkten wie Pharmaproteinen, Albu min-Präparaten oder Heparin existieren nach wie vor große Schwierigkeiten: Treten in diesen Präparaten Endotoxin-Verunrei nigungen auf, bleibt gegenwärtig nur die Möglichkeit des Repro zessierens, um den Vorschriften von FDA, USP oder EP gerecht zu werden.In the case of high molecular weight products such as pharmaceutical proteins, Albu Min supplements or heparin still exist in large quantities Difficulty: Endotoxin contamination occurs in these preparations only the possibility of repro to meet the requirements of FDA, USP or EP will.
Um dieses aufwendige Verfahren zu vermeiden, das Produkt aber trotzdem seiner Bestimmung zuzuführen, wurde die Möglichkeit ge prüft, verseuchte Produkte über chromatographische Sorbentien mit Endotoxin-spezifischen Liganden selektiv zu dekontaminieren. Auch dies brachte nicht den gewünschten Erfolg: Bisher beschrie bene Affinitätssorbentien mit His, Him, PMB als Liganden erwie sen sich trotz hoher bis sehr hoher Assoziationskonstanten bei hohen Endotoxin-Eingangskonzentrationen als nicht geeignet (9). Ferner wurde in Gegenwart von Proteinen konkurrierende Pro teinadsorption beobachtet, die zu verringerten Endotoxin-Abrei cherungsraten und teilweise hohen Proteinverlusten führte (insbesondere bei sauren Proteinen wie BSA).In order to avoid this complex process, the product nevertheless, to lead to its purpose, the possibility was ge checks contaminated products via chromatographic sorbents to decontaminate selectively with endotoxin-specific ligands. This also did not bring the desired success: So far described bene affinity sorbents with His, Him, PMB as ligands despite high to very high association constants high endotoxin concentrations as unsuitable (9). In the presence of proteins, competing Pro Tin adsorption observed, leading to decreased endotoxin abrasion backup rates and sometimes high protein losses (especially with acidic proteins like BSA).
(1) S.K. Sharma
Endotoxin detection and elimination in biotechnology
Biotechnol. Appl. Biochem. 8 (1986), 5-22
(2) N. Haeffner-Cavaillon, J.M. Cavaillon, L. Szabó
Cellular receptors for endotoxin
Handbook of Endotoxins, Vol. 3: Biology of Endotoxins,
1-24
Elsevier Science Publishers B.V. (1985)
(3) D.C. Morrison, J.L. Ryan
Endotoxins and disease mechanisms
Ann. Rev: Med. 38 (1987), 417-32
(4) USP XXII Suppl. 5 (Nov. 1991)
(5) K.C. Hou, R. Zaniewski
Depyrogenation by endotoxin removal with positively
charged depth filter cartridge
J. Parenteral Sci. Tech., Vol. 44, No. 4 (1990), 204-209
(6) CUNO Newsletter for Pharmaceuticals (Okt. 1995), S. 3
(7) B.P. Smollich, D. Falkenhagen, J. Schneidewind, S. Mitzner,
H. Klinkmann
Importance of endotoxins in high-flux dialysis
Nephrol. Dial. Transplant 3 (Suppl.) (1991) 83-85
(8) E. Flindt
Pyrogenentfernung mittels Ultrafiltration
Memoscript CONCEPT-Symposium "Pyrogene II"
(Juni 1983), S. 54-60
(9) F.B. Anspach, O. Hillbeck
Removal of endotoxins by affinity sorbents
J. Chromatogr. A 711 (1995), 81-92(1) SK Sharma
Endotoxin detection and elimination in biotechnology Biotechnol. Appl. Biochem. 8: 5-22 (1986)
(2) N. Haeffner-Cavaillon, JM Cavaillon, L. Szabó Cellular receptors for endotoxin
Handbook of Endotoxins, Vol. 3: Biology of Endotoxins, 1-24
Elsevier Science Publishers BV (1985)
(3) DC Morrison, JL Ryan
Endotoxins and disease mechanisms
Ann. Rev: Med. 38 (1987), 417-32
(4) USP XXII Suppl. 5 (Nov. 1991)
(5) KC Hou, R. Zaniewski
Depyrogenation by endotoxin removal with positively charged depth filter cartridge
J. Parenteral Sci. Tech., Vol. 44, No. 4: 204-209 (1990)
(6) CUNO Newsletter for Pharmaceuticals (Oct. 1995), p. 3
(7) BP Smollich, D. Falkenhagen, J. Schneidewind, S. Mitzner, H. Klinkmann
Importance of endotoxins in high-flux dialysis
Nephrol. Dial. Transplant 3 (Suppl.) (1991) 83-85
(8) E. Flindt
Pyrogen removal using ultrafiltration
Memoscript CONCEPT symposium "Pyrogene II" (June 1983), pp. 54-60
(9) FB Anspach, O. Hillbeck
Removal of endotoxins by affinity sorbents
J. Chromatogr. A 711 (1995), 81-92
Dieser Stand der Technik läßt sich erfindungsgemäß durch eine Mikrofiltrationsmembran zur Abtrennung von Endotoxinen aus flüs sigen Medien, insbesondere Wasser, Proteinlösungen oder Parente ralien verbessern, wobei die Mikrofiltrationsmembran durch kova lent gebundene Liganden für Endotoxine gekennzeichnet ist, wobei die Liganden von einem Polymeren getragen werden, das auf der Membran aufgebracht ist.This prior art can be inventively by Microfiltration membrane for the separation of endotoxins from fluids media, especially water, protein solutions or parents Improve ralia, the microfiltration membrane by kova lent bound ligands for endotoxins, wherein the ligands are carried by a polymer based on the Membrane is applied.
Zur Membrantechnologie und auch Membranherstellung kann auf Ho & Sirkar (Herausgeber), Membrane Handbook, Verlag van Nostrand Reinhold, New York, 1992, verwiesen werden.For membrane technology and membrane production, Ho & Sirkar (Editor), Membrane Handbook, Verlag van Nostrand Reinhold, New York, 1992.
Bei den kovalent gebundenen Liganden kann es sich umThe covalently bound ligands can be
- (a) einen endotoxin-spezifischen Liganden, vorzugsweise Histamin, Histidin, Polyethylenimin, Poly-L-lysin oder Poly myxin B und/oder(a) an endotoxin-specific ligand, preferably Histamine, histidine, polyethyleneimine, poly-L-lysine or poly myxin B and / or
- (b) einen per se nicht-endotoxin-spezifischen Liganden handeln, vorzugsweise Diaminohexan, Diethylaminoethyl oder Desoxycho lat.(b) act as a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycho lat.
Bei dem Membranmaterial für die erfindungsgemäße Mikrofiltra tionsmembran kann es sich um regenerierte Zellulose, Zellulose acet, Polysulfon, Polyethylenvinylalkohol oder Polyamid handeln, insbesondere um Nylon.In the membrane material for the microfiltra according to the invention tion membrane can be regenerated cellulose, cellulose act acet, polysulfone, polyethylene vinyl alcohol or polyamide, especially nylon.
Bei den Polymeren, das auf die erfindungsgemäße Mikrofiltra tionsmembran aufgebracht ist, kann es sich um ein hydrophiles Polymere handeln, insbesondere um Dextran, Polyvinylalkohol oder modifizierte Zellulose, vorzugsweise Hydroxyethylzellulose.In the case of the polymers, the microfiltra according to the invention tion membrane is applied, it can be a hydrophilic Act polymers, in particular dextran, polyvinyl alcohol or modified cellulose, preferably hydroxyethyl cellulose.
Dieses hydrophile Polymere kann für sich wasserlöslich, in Was ser quellbar oder wasserunlöslich sein.This hydrophilic polymer can be water-soluble, in what be swellable or water-insoluble.
Das Polymere kann von der erfindungsgemäßen Mikrofiltrationsmem bran mit Hilfe eines Spacers getragen werden. Auch die kovalent gebundenen Liganden können von einem Spacer getragen werden. Bei diesen Spacern kann es sich um Spacer handeln, die sich von Bisoxiran, Glutardialdehyd, Epihalogenhydrin oder Diisocyanat herleiten, gegebenenfalls nach oxidativer Aktivierung.The polymer can be removed from the microfiltration membrane according to the invention bran can be worn with the help of a spacer. The covalent too bound ligands can be carried by a spacer. At these spacers can be spacers that differ from Bisoxirane, glutaraldehyde, epihalohydrin or diisocyanate derive, if necessary after oxidative activation.
Zur Aktivierungs- und Immobilisierungschemie, die auch auf endo toxin-spezifische Liganden und Spacer eingeht, sei beispiels weise auf Hermanson, Mallia & Smith, Immobilized Affinity Ligand Techniques, Academic Press Inc., San Diego, 1992, verwiesen.For activation and immobilization chemistry, which is also available on endo toxin-specific ligands and spacers, for example point to Hermanson, Mallia & Smith, Immobilized Affinity Ligand Techniques, Academic Press Inc., San Diego, 1992.
Erfindungsgemäß werden also Mikrofiltrationsmembranen vorgese hen, die in geeigneter Weise oberflächenmodifiziert sind und aus Wasser und wäßrigen Lösungen (Puffer, Proteinlösungen) Endoto xine entfernen. Die Oberflächenmodifikation kann im Aufbringen eines bifunktionellen kovalent gebundenen Spacers bestehen, der mit einem hydrophilen Polymeren umgesetzt wird, wobei unspezifi sche Wechselwirkungen der Membran, speziell mit Proteinen, redu ziert werden. Das kovalent gebundene hydrophile Polymere kann mit endotoxin-spezifischen Liganden umgesetzt werden, gegebenen falls über einen weiteren Spacer. Zum Prinzip der Oberflächenmo difikation der Membran vergleiche man Fig. 1.According to the invention, microfiltration membranes are provided, which are surface-modified in a suitable manner and remove endotoxins from water and aqueous solutions (buffers, protein solutions). The surface modification can consist in the application of a bifunctional covalently bonded spacer which is reacted with a hydrophilic polymer, non-specific interactions of the membrane, especially with proteins, being reduced. The covalently bound hydrophilic polymer can be reacted with endotoxin-specific ligands, if necessary via a further spacer. For the principle of surface modification of the membrane, see FIG. 1.
Der Endotoxin-Abreicherungserfolg kann sich in Gegenwart von Proteinen als abhängig von der Nettoladung der Proteine erwei sen. Durch Optimierung der Bedingungen (pH-Wert) können saure Proteine (wie BSA und Maus-IgG 1) vollständig dekontaminiert werden, und zwar ohne nennenswerte Verluste an Protein. Im Fall basischer Proteine (wie beispielsweise Lysozym und bFGF) lassen sich ebenfalls hohe Abreicherungen erzielen.Endotoxin depletion success may vary in the presence of Proteins as dependent on the net charge of the proteins sen. By optimizing the conditions (pH value) acidic Proteins (such as BSA and mouse IgG 1) completely decontaminated without any significant loss of protein. In the case basic proteins (such as lysozyme and bFGF) high depletions are also achieved.
Polymer-gecoatete erfindungsgemäße Mikrofiltrationsmembranen mit kovalent gebundenen endotoxin-spezifischen Liganden können Endo toxine in einem Durchgang entfernen, und zwar auch aus hochbela steten Lösungen (6000 EU ml-1).Polymer-coated microfiltration membranes according to the invention with covalently bound endotoxin-specific ligands can remove endotoxins in one pass, even from highly loaded solutions (6000 EU ml -1 ).
Vom Prinzip her sind die Membranen entsprechend Fig. 1. aufge baut. Zunächst wird über einen Spacer ein hydrophiles Polymer aufgebracht, das dann weiter, ggf. über einen Spacer, mit Endo toxin-spezifischen Liganden umgesetzt wird. Als Membranmateria lien kommen insbesondere infrage:In principle, the membranes are built up according to Fig. 1. First, a hydrophilic polymer is applied via a spacer, which is then further, optionally via a spacer, reacted with endotoxin-specific ligands. The following are particularly suitable as membrane materials:
- - Cellulose- cellulose
- - Polysulfon- polysulfone
- - PEVA (Polyethylenvinylalkohol)- PEVA (polyethylene vinyl alcohol)
- - Polyamide (speziell Nylon, wie z. B. N66)- polyamides (especially nylon, e.g. N66)
Als Spacer eignen sich reaktive bifunktionale Verbindungen. Spe ziell geeignet sind:Reactive bifunctional compounds are suitable as spacers. Spe the following are suitable:
- - BisoxiranBisoxirane
- - Glutardialdehyd- glutardialdehyde
- - Epihalogenhydrine- epihalohydrins
- - Diisocyanate- diisocyanates
Zur Aktivierung der bei Verwendung von Bisoxiran und Epihalogen hydrin entstehenden vicinalen Diolbindung kann ggf. Oxidation durch Periodat angewandt werden, wobei eine Aldehydgruppe ent steht. Der an die Membran gebundene Spacer wird weiter umgesetzt mit hydrophilen Polymeren. Als solche kommen bevorzugt infrage:To activate when using bisoxiran and epihalogen The vicinal diol bond formed in hydrine can possibly oxidize be applied by periodate, with an aldehyde group ent stands. The spacer bound to the membrane is further implemented with hydrophilic polymers. As such, the following are preferred:
- - Dextran- dextran
- - Polyvinylalkohol (PVA)- polyvinyl alcohol (PVA)
- - Modifizierte Cellulosen, speziell Hydroxyethylcellulose (HEC)- Modified celluloses, especially hydroxyethyl cellulose (HEC)
Die weitere Reaktion erfolgt entweder direkt mit dem Endotoxin spezifischen Liganden oder wiederum über die Vermittlung eines der oben angeführten Spacer, ggf. nach dessen oxidativer Akti vierung. Als Endotoxin-spezifische Liganden wirken (s. Liste der Abkürzungen): DAH, Him, His, PEI, PLL, PMB. Auch die üblicher weise nicht endotoxin-spezifischen Liganden wie DEAE und DOC er wiesen sich in der Membrankonfiguration als hochspezifisch bei gleichzeitig hoher Wiederfindung der Proteine.The further reaction takes place either directly with the endotoxin specific ligands or again through the mediation of a the above-mentioned spacer, if necessary after its oxidative acti crossing. Act as endotoxin-specific ligands (see list of Abbreviations): DAH, Him, His, PEI, PLL, PMB. Even the more common have non-endotoxin-specific ligands such as DEAE and DOC proved to be highly specific in the membrane configuration at the same time high recovery of the proteins.
Die Leistungsfähigkeit der entwickelten Membranen geht aus den angeführten Beispielen hervor. Die Endotoxin-Entfernung kann fast immer als vollständig bezeichnet werden. Sie liegt in der Regel unter 1 EU ml-1, oft unterhalb der Nachweisgrenze mit dem LAL-Test.The performance of the developed membranes can be seen from the examples given. Endotoxin removal can almost always be said to be complete. It is usually below 1 EU ml -1 , often below the detection limit with the LAL test.
An Kontrollmembranen (Nylon ohne Modifikation und mit aufge brachtem hydrophoben Polymer mit oder ohne Spacer) ohne endoto xin-spezifischen Liganden wurde keine Endotoxinabreicherung er halten.On control membranes (nylon without modification and with attached brought hydrophobic polymer with or without spacer) without endoto xin-specific ligands, no endotoxin depletion hold.
Die neuen Membranen können eingesetzt werden zur Endotoxinent fernung aus Wasser und Parenteralia. Auch in Gegenwart von Pro teinen werden gute Ergebnisse erzielt. Im Fall basischer Pro teine ist allerdings zu berücksichtigen, daß Wechselwirkungen der Proteine mit dem Endotoxin auftreten können, die zu einer endotoxischen Maskierung führen können. Proteingebundenes Endo toxin kann mit dem LAL-Test nicht eindeutig nachgewiesen werden. In diesem Zusammenhang ist zu erwähnen, daß nicht endgültig ge klärt ist, ob proteingebundenes Endotoxin noch toxisch wirkt.The new membranes can be used as an endotoxin Removal from water and parenterals. Even in the presence of Pro good results are achieved. In the case of basic pro However, one must take into account that interactions of the proteins with the endotoxin that can lead to a endotoxic masking. Protein-bound endo toxin cannot be clearly demonstrated with the LAL test. In this context it should be mentioned that ge It is clear whether protein-bound endotoxin is still toxic.
Die erfindungsgemäßen Membranen können vielseitig angewandt wer den.The membranes of the invention can be used in a variety of ways the.
Medizinisch-pharmazeutischer Bereich:Medical-pharmaceutical area:
- - Hämodialyse.- hemodialysis.
- - Unbedenkliche Infusions- und Injektionslösungen (Parenteralia)- Safe infusion and injection solutions (Parenterals)
- - Sichere Diagnostika (z. B. Antikörper).- Safe diagnostics (e.g. antibodies).
In der Biotechnologie:In biotechnology:
- - Herstellung von Pharmaprodukten.- Manufacture of pharmaceutical products.
- - Endotoxin-Abreicherung in Prozeßwasser und Rohstoffen.- Endotoxin depletion in process water and raw materials.
- - Dekontaminierung von Produkten (Aufwand für Prozessierung ent fällt)- Decontamination of products (expenditure for processing ent falls)
An Mikrofiltrationsmembranen auf Nylon-Basis (bevorzugt 0,45 µm oder größer) werden hydrophile Polymere, insbesondere Dextran, Polyvinylalkohol und Hydroxyethylcellulose kovalent gebunden. Im nächsten Schritt werden an die aufgebrachten Polymere endotoxin spezifische Liganden immobilisiert. Fig. 1 illustriert den Auf bau der Membranen.Hydrophilic polymers, in particular dextran, polyvinyl alcohol and hydroxyethyl cellulose, are covalently bound to microfiltration membranes based on nylon (preferably 0.45 μm or larger). In the next step, endotoxin-specific ligands are immobilized on the applied polymers. Fig. 1 illustrates the construction of the membranes.
Die Nylon-Membranen wurden zunächst mit Bisoxiran aktiviert:
Hierzu wurden sie sechzehn Stunden bei 80°C in einer Mischung
von 9 ml Bisoxiran, 1 ml Ethanol und 1 ml 25 mM Natriumcarbo
nat-Puffer (pH 11) geschüttelt (Fig. 2a). Nach gründlichem Waschen
wurde je eine Membran mit 5 ml einer 20-proz. Dextran 40 000-Lö
sung (pH 11) fünfzehn Minuten bei Raumtemperatur inkubiert
(Fig. 2b). Anschließend wurden die Membranen 14 Stunden bei 120°C
getrocknet. Um unspezifisch gebundenes Dextran zu entfernen,
wurden die Membranen dreimal mit 0,1 M Natronlauge und weitere
dreimal mit Wasser gewaschen.The nylon membranes were first activated with bisoxirane:
For this purpose, they were shaken for sixteen hours at 80 ° C. in a mixture of 9 ml bisoxirane, 1 ml ethanol and 1 ml 25 mM sodium carbonate buffer (pH 11) ( FIG. 2a). After thorough washing, each membrane with 5 ml of a 20 percent. Dextran 40,000 solution (pH 11) incubated for fifteen minutes at room temperature ( Fig. 2b). The membranes were then dried at 120 ° C. for 14 hours. To remove non-specifically bound dextran, the membranes were washed three times with 0.1 M sodium hydroxide solution and three more times with water.
Wie Fig. 3 zeigt, zeichnen sich die gecoateten Membranen durch signifikant geringere unspezifische Wechselwirkungen - ausge drückt durch adsorbierte Menge Hämoglobin - aus.As shown in Fig. 3, the coated membranes are characterized by significantly lower non-specific interactions - expressed by the amount of hemoglobin adsorbed.
Aus der Fig. 3 geht auch hervor, daß mit einem einfachen Dextran-coating nicht der gleiche Effekt erreicht werden kann wie mit PVA und HEC. Durch einen zweiten Layer kann weitere Ver besserung erzielt werden, während ein dritter Layer nur noch ge ringfügige Auswirkungen hat. Dextran wurde daher immer als Dop pel-coating eingesetzt.From Fig. 3 also shows that with a simple dextran coating, the same effect can not be achieved as with PVA and HEC. A second layer can be used for further improvement, while a third layer has only minor effects. Dextran was therefore always used as a double coating.
Die Liganden PLL, PMB und PEI wurden entweder direkt an die periodat-aktivierten Coating-Polymere oder nach Inkorporation eines periodat-oxidierbaren Spacers (Bisoxiran) immobilisiert. Das Vorgehen ist beispielhaft in Fig. 4 dargestellt. DEAE wurde ohne Spacer direkt an die Matrices gekoppelt, die anderen nie dermolekularen Liganden wurden über Epibromhydrin gebunden.The ligands PLL, PMB and PEI were either immobilized directly on the periodate-activated coating polymers or after incorporation of a periodate-oxidizable spacer (bisoxirane). The procedure is shown by way of example in FIG. 4. DEAE was coupled directly to the matrices without a spacer, the other never dermolecular ligands were bound via epibromohydrin.
Zur Aktivierung wurden die mit hydrophilen Polymeren gecoateten Membranen drei Stunden bei Raumtemperatur in einer Mischung aus 100 mg Natriumborhydrid, 5 ml Bisoxiran und 45 ml 1 M Natron lauge inkubiert. Nach Hydrolyse des freien Oxiranringes (30 Mi nuten Inkubation bei pH 2,5) und Periodatoxidation des erhalte nen vicinalen Diols (90 Minuten Inkubation in 0,2 M Natrium periodat) wurden die Membranen zwei Stunden mit einer Lösung von 0,5 g PEI (MW 50 000) in 0,1 M Phosphatpuffer, die auf pH 8 ein gestellt wurde, bei Raumtemperatur umgesetzt, so daß sich der in Fig. 1 gezeigte Aufbau ergibt. Abschließend wurde mit 1 M Na triumchlorid-Lösung und Wasser gewaschen.For activation, the membranes coated with hydrophilic polymers were incubated for three hours at room temperature in a mixture of 100 mg sodium borohydride, 5 ml bisoxirane and 45 ml 1 M sodium hydroxide solution. After hydrolysis of the free oxirane ring (30 minutes incubation at pH 2.5) and periodate oxidation of the vicinal diol obtained (90 minutes incubation in 0.2 M sodium periodate), the membranes were washed with a solution of 0.5 g PEI ( MW 50,000) in 0.1 M phosphate buffer, which was adjusted to pH 8, at room temperature, so that the structure shown in FIG. 1 results. Finally, it was washed with 1 M sodium chloride solution and water.
Histidin wurde über DAH an epibromhydrin-aktivierte gecoatete Membranen immobilisiert. Epibromhydrin-Aktivierung wurde wie für Bisoxiran beschrieben durchgeführt. Immobilisiertes DAH wurde durch 8-minütige Reaktion mit einer Mischung von 5 ml Epibromhy drin und 5 ml 4 M Natronlauge bei 90°C aktiviert und sofort bei 80°C mit L-Histidin umgesetzt (0,5 g L-Histidin in 20 ml Was ser, pH 12). Die fertige Membran wurde mit 1 M Natriumchlorid und Wasser gewaschen.Histidine was coated on epibromohydrin-activated via DAH Immobilized membranes. Epibromohydrin activation was like for Bisoxiran described performed. DAH was immobilized by reaction for 8 minutes with a mixture of 5 ml of Epibromhy activated in it and 5 ml of 4 M sodium hydroxide solution at 90 ° C and immediately at 80 ° C with L-histidine (0.5 g L-histidine in 20 ml Was water, pH 12). The finished membrane was covered with 1 M sodium chloride and washed water.
Entsprechende Vorschriften wurden angewandt für das Coating mit anderen Polymeren und die kovalente Bindung mit den anderen en dotoxin-spezifischen Liganden.Corresponding regulations were applied for the coating with other polymers and the covalent bond with the other en dotoxin-specific ligands.
Alle Untersuchungen zum Adsorptionsverhalten von Endotoxinen an den Membranen wurden bei Raumtemperatur im Dead-end-Modus durch geführt.All studies on the adsorption behavior of endotoxins the membranes were run through at room temperature in dead-end mode guided.
Je eine Membranscheibe wurde am Boden einer Ultrafiltrations zelle (Membranfläche 13,4 cm²) fixiert und mit 30% ethanoli scher 0,1 M Natronlauge, 1,5 M Natriumchlorid-Lösung und pyro genfreiem Wasser gewaschen, um Endotoxin-Spuren zu entfernen. Nach Equilibrierung der Membran wurden jeweils 20 ml kontami nierter Lösung mit einer Flußgeschwindigkeit von 2 ml/min durch die Membran filtriert. Das Filtrat wurde gesammelt und im LAL-Test untersucht. One membrane disk was placed on the bottom of an ultrafiltration cell (membrane area 13.4 cm²) fixed and with 30% ethanoli 0.1 M sodium hydroxide solution, 1.5 M sodium chloride solution and pyro GM free water washed to remove traces of endotoxin. After equilibration of the membrane, 20 ml each were contaminated nated solution at a flow rate of 2 ml / min the membrane is filtered. The filtrate was collected and in the LAL test examined.
Zur Endotoxin-Quantifizierung in den Ausgangslösungen und Filtraten wurde ein chromogener Limulus Amöbozyt Lysat Test (LAL-Test) eingesetzt. Dieser Test beruht darauf, daß Endotoxin die Freisetzung des Chromogens p-Nitroanilin induziert, wobei zwischen der freigesetzten Menge p-Nitroanilin und der vorlie genden Endotoxin-Konzentration im Bereich 0 bis 1,2 EU/ml ein linearer Zusammenhang besteht. Aus der photometrischen p-Nitro anilin-Bestimmung kann mit Hilfe einer Kalibriergeraden (Standard-Endotoxin E. coli 0111 : B4) auf die Endotoxin-Konzen tration der Proben geschlossen werden.For endotoxin quantification in the starting solutions and A chromogenic Limulus amebocyte lysate test was used to filter the filtrates (LAL test) used. This test is based on endotoxin induces the release of the chromogen p-nitroaniline, whereby between the released amount of p-nitroaniline and the present endotoxin concentration in the range 0 to 1.2 EU / ml linear relationship exists. From the photometric p-nitro Aniline determination can be done using a calibration line (Standard endotoxin E. coli 0111: B4) on the endotoxin concentrations tration of the samples can be closed.
Der LAL-Test wurde in Europa 1985 von der Europäischen Arznei buch-Kommission zur Prüfung auf Endotoxine eingeführt und er setzt seit 1989 auch in der Monographie "Wasser für Injektions zwecke" den Kaninchen-Test.The LAL test was launched in Europe in 1985 by the European Medicines book commission for testing for endotoxins and he introduced has been using water for injections in the monograph since 1989 purpose "the rabbit test.
1. (Fig. 5) Abreicherung aus hochbelasteten Pufferlösungen
Feed: 20 ml 20 mM Phosphatpuffer (pH 7) mit 6000 EU/ml
versetzt
Die mit -d gekennzeichneten Membranen stellen Membranen dar,
bei denen auf Inkorporation eines Spacers verzichtet wurde.1. ( Fig. 5) depletion from highly loaded buffer solutions Feed: 20 ml 20 mM phosphate buffer (pH 7) mixed with 6000 EU / ml
The membranes marked with -d represent membranes in which the incorporation of a spacer has been dispensed with.
2. (Fig. 6 bis 7) Abreicherung aus Endotoxin-angereicherten
BSA-Lösungen
Feed: 20 ml 20 mM Phosphatpuffer (pH 4,66) mit 1 mg/ml BSA
und 6610 EU/ml versetzt
Proteinwiederfindung BSA
2. ( Fig. 6 to 7) depletion from endotoxin-enriched BSA solutions
Feed: 20 ml 20 mM phosphate buffer (pH 4.66) mixed with 1 mg / ml BSA and 6610 EU / ml
Protein recovery BSA
3. (Fig. 8) Abreicherung aus Handels-BSA
Feed: 20 ml 20 mM Phosphatpuffer (pH 4,66) mit 1 mg/ml BSA
Endotoxinkonzentration 65 EU/ml3. ( Fig. 8) depletion from commercial BSA
Feed: 20 ml 20 mM phosphate buffer (pH 4.66) with 1 mg / ml BSA
Endotoxin concentration 65 EU / ml
4. (Fig. 9 bis 10) Abreicherung aus Handels-Lysozym
Feed: 20 ml 20 mM Phosphatpuffer (pH 7) mit 1 mg/ml Lysozym
Endotoxinkonzentration 134 EU/ml
Proteinwiederfindung Lysozym4. ( Fig. 9 to 10) depletion from commercial lysozyme
Feed: 20 ml 20 mM phosphate buffer (pH 7) with 1 mg / ml lysozyme endotoxin concentration 134 EU / ml
Protein recovery lysozyme
5. (Fig. 11 bis 12) Abreicherung aus MAX 16 H 5
Feed: 20 ml 20 mM Phosphatpuffer (pH 5,5) mit 3 mg/ml Protein
Endotoxinkonzentration 62,5 EU/ml
Proteinwiederfindung IgG5. ( Fig. 11 to 12) depletion from MAX 16 H 5
Feed: 20 ml 20 mM phosphate buffer (pH 5.5) with 3 mg / ml protein
Endotoxin concentration 62.5 EU / ml
Protein recovery IgG
6. Abreicherung aus bereits aufgereinigtem bFGF
Feed: 5 ml bFGF, das 9 EU/ml enthielt
Es wurde das Abreicherungsverfahren einer PEI-Membran unter
sucht. Im Filtrat waren noch 0,202 EU/ml nachweisbar.6. Depletion from already purified bFGF
Feed: 5 ml bFGF, which contained 9 EU / ml
The depletion process of a PEI membrane was examined. 0.202 EU / ml was still detectable in the filtrate.
7. Abreicherung aus Milli-Q-Wasser, das mit Endotoxin versetzt
wurde
Feed: 1 l Wasser mit 270 EU/ml versetzt
Zur Abreicherung wurden eine PEI- und eine DAHHis-Membran
eingesetzt.
PEI-Filtrat: < 0,015 EU/ml
DAHHis-Filtrat: 0,07 EU/ml
7. Depletion from Milli-Q water that has been treated with endotoxin
Feed: 1 l water mixed with 270 EU / ml
A PEI and a DAHHis membrane were used for depletion.
PEI filtrate: <0.015 EU / ml
DAHHis filtrate: 0.07 EU / ml
BSA Rinderserumalbumin
bFGF basischer fibroblasten-Wachstumsfaktor
DAH Diaminohexan
DEAE Diethyl-aminoethyl
DEX Dextran
DEX/2 bezeichnet eine Membran, die zweimal nacheinander mit Dextran gecoatet
wurde
DEX/3 bezeichnet eine Membran, die dreimal nacheinander mit Dextran gecoatet
wurde
DOC Desoxycholat
EP Europäische Pharmacopö
EU Endotoxin-Unit
FDA Food and drug administration
HEC Hydroxyethylcellulose
Him Histamin
His Histidin
MW Molekulargewicht
N66 unbehandelte Nylon-Membran
PEI Polyethylenimin
PLL Poly-L-Lysin
PMB Polymyxin B
PVA Polyvinylalkohol
USP US PharmacopöBSA bovine serum albumin
bFGF basic fibroblast growth factor
DAH diaminohexane
DEAE diethylaminoethyl
DEX dextran
DEX / 2 is a membrane that has been coated twice in succession with dextran
DEX / 3 is a membrane that has been coated three times in a row with dextran
DOC deoxycholate
EP European Pharmacopoeia
EU endotoxin unit
FDA Food and drug administration
HEC hydroxyethyl cellulose
Him histamine
His histidine
MW molecular weight
N66 untreated nylon membrane
PEI polyethyleneimine
PLL poly-L-lysine
PMB polymyxin B
PVA polyvinyl alcohol
USP US Pharmacopoeia
Claims (10)
- (a) einen endotoxin-spezifischen Liganden, vorzugsweise Histamin, Histidin, Polyethylenimin, Poly-L-lysin oder Polymyxin B und/oder
- (b) einen per se nicht-endotoxin-spezifischen Liganden, vorzugs weise Diaminohexan, Diethylaminoethyl oder Desoxycholat.
- (a) an endotoxin-specific ligand, preferably histamine, histidine, polyethyleneimine, poly-L-lysine or polymyxin B and / or
- (b) a non-endotoxin-specific ligand per se, preferably diaminohexane, diethylaminoethyl or deoxycholate.
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DE19609479A DE19609479A1 (en) | 1996-03-11 | 1996-03-11 | Endotoxin-specific membranes |
CA002249548A CA2249548A1 (en) | 1996-03-11 | 1997-03-11 | Endotoxin-specific membranes |
EP97914219A EP0888172A1 (en) | 1996-03-11 | 1997-03-11 | Endotoxin-specific membranes |
PCT/EP1997/001225 WO1997033683A1 (en) | 1996-03-11 | 1997-03-11 | Endotoxin-specific membranes |
JP53227797A JP2002514970A (en) | 1996-03-11 | 1997-03-11 | Endotoxin-specific membrane |
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JP (1) | JP2002514970A (en) |
CA (1) | CA2249548A1 (en) |
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WO (1) | WO1997033683A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000016897A1 (en) * | 1997-09-17 | 2000-03-30 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Microfiltration filter layer for separating endotoxins and the use of said microfiltration filter layer |
WO2001023413A1 (en) * | 1999-09-29 | 2001-04-05 | Gambro Dialysatoren Gmbh & Co., Kg | Extracorporeal endotoxin removal method |
WO2003097112A1 (en) * | 2002-05-22 | 2003-11-27 | Prometic Biosciences Ltd. | Endotoxin-binding ligands and their use |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6780327B1 (en) | 1999-02-25 | 2004-08-24 | Pall Corporation | Positively charged membrane |
FR2902670B1 (en) | 2006-06-22 | 2009-04-24 | Gambro Lundia Ab | USE OF A SUSPENSION FOR TREATING MEDICAL MEDIA, MEDICAL MEDIA, EXCHANGER, AND ADSORPTION DEVICE COMPRISING THE MEDIUM |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB2092470B (en) * | 1981-02-10 | 1984-07-18 | Tanabe Seiyaku Co | Method for reducing the pyrogen content of or removing pyrogens from solutions contaminated therewith |
JPH0829316B2 (en) * | 1987-10-16 | 1996-03-27 | 田辺製薬株式会社 | How to remove Pyrogen |
US5032281A (en) * | 1989-08-09 | 1991-07-16 | Daicel Chemical Industries, Ltd. | Separating membrane and separation method |
DE4209988A1 (en) * | 1991-04-23 | 1993-03-04 | Falkenhagen Dieter Dr Sc Med | Endotoxin adsorber having high binding capacity - comprises polyethyleneimine bonded to porous carrier, esp. polysaccharide |
-
1996
- 1996-03-11 DE DE19609479A patent/DE19609479A1/en not_active Withdrawn
-
1997
- 1997-03-11 EP EP97914219A patent/EP0888172A1/en not_active Withdrawn
- 1997-03-11 CA CA002249548A patent/CA2249548A1/en not_active Abandoned
- 1997-03-11 WO PCT/EP1997/001225 patent/WO1997033683A1/en not_active Application Discontinuation
- 1997-03-11 JP JP53227797A patent/JP2002514970A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000016897A1 (en) * | 1997-09-17 | 2000-03-30 | GESELLSCHAFT FüR BIOTECHNOLOGISCHE FORSCHUNG MBH (GBF) | Microfiltration filter layer for separating endotoxins and the use of said microfiltration filter layer |
WO2001023413A1 (en) * | 1999-09-29 | 2001-04-05 | Gambro Dialysatoren Gmbh & Co., Kg | Extracorporeal endotoxin removal method |
WO2003097112A1 (en) * | 2002-05-22 | 2003-11-27 | Prometic Biosciences Ltd. | Endotoxin-binding ligands and their use |
CN1327901C (en) * | 2002-05-22 | 2007-07-25 | 普罗米蒂克生物科学有限公司 | Endotoxin-binding ligands and their use |
US8765393B2 (en) | 2002-05-22 | 2014-07-01 | Prometic Biosciences Ltd | Endotoxin-binding ligands and their use |
Also Published As
Publication number | Publication date |
---|---|
WO1997033683A1 (en) | 1997-09-18 |
CA2249548A1 (en) | 1997-09-18 |
JP2002514970A (en) | 2002-05-21 |
EP0888172A1 (en) | 1999-01-07 |
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